Lecture 1 Glycolysis PDF

UNIVERSITY OF LUSAKA School of Medicine and Health Sciences Department of Basic Sciences Biochemistry II BMPS 111 LECTURE 1 GLYCOLYSIS  USEFUL INFORMATION!! Assessment plan for BMPS 111 - The final mark wil...

UNIVERSITY OF LUSAKA School of Medicine and Health Sciences Department of Basic Sciences Biochemistry II BMPS 111 LECTURE 1 GLYCOLYSIS  USEFUL INFORMATION!! Assessment plan for BMPS 111 - The final mark will be calculated as follows: 40% Continuous Assessment + 60% exam mark - The semester mark is calculated as follows: - 1 Group Assignment (Oral presentation) - Assignment will be given – Friday 12th August - Due Friday Sept 9th (Mini Review) - Oral presentation – TBA - Mid Semester Exam - 2 Tests - THERE WILL BE NO DEFERRED TESTS CONDUCTED.  Learning Outcomes 1. Describe the main stages of glycolysis 2. Understand the fates of pyruvate 3. Understand that ATP formation is coupled to glycolysis 4. Understand the importance of phosphorylated intermediates 5. Understand the net gain of ATP during glycolysis  Cellular respiration: An overview  Cellular respiration - a set of metabolic reactions and processes that take place in the cells of organisms to convert chemical energy from oxygen molecules or nutrients into adenosine triphosphate (ATP), and then release waste products.  Two types of cellular respiration: - Anaerobic and aerobic  Anaerobic - Anaerobic (cellular) respiration is a respiratory process that occurs in both prokaryotes and eukaryotes in which cells break down the sugar molecules to produce energy without the presence of oxygen  Cellular respiration: An overview  Aerobic respiration - requires oxygen (O2) in order to create ATP.  It is the preferred method of pyruvate breakdown in glycolysis, and requires pyruvate to the mitochondria in order to be fully oxidized by the citric acid cycle.  There are three major stages in aerobic respiration. 1. Glycolysis 2. The Krebs Cycle (Citric /Tricarboxcylic Acid  GLYCOLYSIS  The glycolytic pathway is a pathway used by all tissues for the breakdown of glucose.  Glucose is metabolized to pyruvate by the pathway of glycolysis, which occurs aerobically.  When it occurs anaerobically, the end product is lactate.  The breakdown of the six-carbon glucose into two molecules of the three- carbon pyruvate occurs in ten steps, the first five of which constitute the preparatory phase. (Figure 1)  Aerobic tissues metabolize pyruvate to acetyl-CoA, which can enter the citric acid cycle for complete oxidation to CO2 and H2O, linked to the formation of ATP in the process of oxidative phosphorylation.  Glucose is the major fuel of most tissues.  Reactions of Glycolytic Pathway  All reactions occur in the cytoplasm.  The conversion of glucose to pyruvate occur in two stages: 1. The first being the preparatory stage (priming) and 2. The second being energy generation stage (pay-off).  The priming stage corresponds to energy investment in the form of ATP where glucose and other hexose sugars are converted to the common product called glyceraldehyde-3-phosphate (GA-3-P).  In the energy generation state, a net of two ATP molecules are formed per glucose molecule metabolised.  In addition to ATP, two molecules of NADH + H+ are formed if the end product is pyruvate.  If the end product is lactate, then NADH + H+ are converted to NAD+.  Importance of phosphorylation in glycolysis  The phosphorylation of a protein can make it active or inactive. Phosphorylation can either activate a protein or inactivate it.  A kinase is an enzyme that phosphorylates proteins.  A phosphatase is an enzyme that dephosphorylates proteins, effectively undoing the action of kinase.  Phosphorylation of glucose serves three important purposes; First, the addition of a phosphate group to glucose effectively traps it in the cell, as G6P cannot diffuse across the lipid bilayer.  Second, the reaction decreases the concentration of free glucose, favouring additional import of the molecule.  Third, the reaction activates the glucose molecule for the glycolytic pathway. Figure 1: Preparatory phase of glycolysis. Two molecules of glyceraldehyde 3-phosphate (GALP) are formed; both pass through the payoff phase. The numbered reaction steps are catalyzed by the enzymes listed on the right. Reactions of Glycolytic Pathway 1. Phosphorylation of glucose:  In this reaction, glucose is first phosphorylated at the hydroxyl group on C-6 and thus glucose 6-phosphate thus formed in the presence of ATP.  The reaction is catalyzed by the specific enzyme glucokinase in liver cells and by non-specific Hexokinase in liver and extrahepatic tissues.  Glucokinase activity in hepatocytes is also increased by insulin.  As blood glucose levels rise following a meal, the cells of the pancreas are stimulated to release insulin into the portal circulation. Glucose + ATP Glu 6-P + ADP Reactions of Glycolytic Pathway 2. Isomerization of glucose 6-phosphate:  The isomerization of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphohexose isomerase.  The reaction is readily reversible and is not a rate-limiting or regulated step. Glu 6-P Fru 6-P Reactions of Glycolytic Pathway 3. Phosphorylation of fructose 6-phosphate:  The irreversible phosphorylation reaction catalyzed by phosphofructokinase 1(PFK-1) is the most important control point and the rate-limiting step of glycolysis.  PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate.  Fructose 2,6-bisphosphate activates PFK-1 of glycolysis.  Fructose 6-phosphate is phosphorylated to Fructose 1,6-bisphosphate through an ATP input. Fru 6-P + ATP Fru 1,6-bisp + ADP Reactions of Glycolytic Pathway 4. Cleavage of Fructose 1,6-bisphosphate:  Fructose 1, 6-bisphosphate is split by the enzyme aldolase into two molecules of triose phosphates:  An aldotriose, glyceraldehyde 3-phosphate (GALP) and  A Ketotriose, Dihydroxyacetone phosphate (DHAP). Fru 1,6-bisp GAP + DHAP Reactions of Glycolytic Pathway 5. Isomerization of dihydroxyacetone phosphate:  Triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.  Dihydroxyacetone phosphate must be isomerized to glyceraldehyde- 3-phosphate for further metabolism by the glycolytic pathway.  This isomerization results in the net production of two molecules of glyceraldehyde 3-phosphate from the cleavage products of fructose 1,6-bisphosphate. DHAP GAP  The Payoff Phase of Glycolysis Yields ATP and NADH Figure 2: Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. The numbered reaction steps are catalyzed by the enzymes  What is NAD?  During glycolysis, NAD( Nicotinamide adenine dinucleotide) Plays a pivotal role.  When it’s oxidised form NAD+ is converted into its reduced form NADH), two molecules of ATP are produced. Reactions of Glycolytic Pathway 6. Oxidation of glyceraldehyde 3- phosphate:  The conversion of glyceraldehyde 3- phosphate to 1,3- bisphosphoglycerate (1,3-BPG) by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation- reduction reaction of glycolysis.  The reaction is an oxidative phosphorylation. 2GALP + 2NAD + 2Pi 2 (1,3-BPG) + 2NADH + 2H+  Reactions of Glycolytic Pathway 7. Synthesis of 3-phosphoglycerate producing ATP:  1,3-BPG is converted to 3-phosphoglycerate (3- PG), the high-energy phosphate group of 1,3-BPG is used to synthesize ATP from ADP.  This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible.  Two molecules of 1,3-BPG are formed from each glucose molecule, this kinase reaction replaces the two ATP molecules consumed by the earlier formation of glucose 6-phosphate and fructose 6- Phosphate. 2 1,3-BPG + 2ADP 2 3-PG + 2ATP Reactions of Glycolytic Pathway 8. Shift of the phosphate group from carbon 3 to carbon 2:  The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible 2 3-PG 2 2-PG  Reactions of Glycolytic Pathway 9. Dehydration of 2-PG:  The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the 2-phosphoglycerate molecule, resulting in the formation of phosphoenolpyruvate (PEP), which contains a high-energy phosphate plus water.  The reaction is reversible despite the high-energy nature of the product. 2 (2-PG) 2(PEP) + 2H2O Reactions of Glycolytic Pathway 10. Formation of pyruvate producing ATP:  The conversion of PEP to pyruvate is catalyzed by pyruvate kinase, the third irreversible reaction of glycolysis.  The equilibrium of the pyruvate kinase reaction favours the formation of ATP.  This is the third irreversible reaction of the glycolytic pathway. 2 PEP + 2 ADP 2 Pyruvate + 2 ATP Reactions of Glycolytic Pathway  Under anaerobic conditions, pyruvate is converted to lactate through a redox reaction.  The reaction helps to yield NAD+.  The enzyme Lactate dehydrogenase catalyses this reversible reaction.  Pyruvate + NADH + H+ Lactate + NAD+  Products of glycolysis  4 molecules of ATP (2 used for activation)  2 Molecules of pyruvate  2 Molecules of NADH  NADH enters ETC to yield 3 ATP from each NADH  Energy Yield of Glycolysis  We can now construct a balance sheet for glycolysis to account for; (1) the fate of the carbon skeleton of glucose, (2) the input of Pi and ADP and the output of ATP, and (3) the pathway of electrons in the oxidation-reduction reactions.  The left-hand side of the following equation shows all the inputs of ATP, NAD+, ADP, and Pi and the right-hand side shows all the outputs (keep in mind that each molecule of glucose yields two molecules of pyruvate)  Cancelling out common terms on both sides of the equation gives the overall equation for glycolysis under aerobic conditions:  Energy Yield of Glycolysis  On-going aerobic glycolysis requires the oxidation of most of this NADH by the electron transport chain, producing approximately three ATP for each NADH molecule entering the chain.  Therefore aerobic glycolysis results in the production of a total of 8 ATP molecules.  Despite the production of some ATP during glycolysis, the end products, pyruvate or lactate, still contain most of the energy originally contained in glucose.  The Tricarboxylic Acid (TCA) cycle is required to release that energy completely. Fates of pyruvate  The Fates of Pyruvate  Pyruvic acid made from glucose through glycolysis, can be converted back to carbohydrates (such as glucose) via gluconeogenesis, or to fatty acids through acetyl-CoA.  Pyruvic acid supplies energy to living cells through the citric acid cycle (also known as the Krebs cycle) when oxygen is present (aerobic respiration); when oxygen is lacking, it ferments to produce lactic acid or alcohol.  This is done in one of two ways; 1. Pyruvate is converted into acetyl- coenzyme A, which is the main input for a series of reactions known as the Krebs cycle.  The net reaction of converting pyruvate into acetyl CoA and CO2 is:  The Fates of Pyruvate  2. (Fermentation) In the absence of oxygen, the acid is broken down anaerobically, creating lactate in animals and ethanol in plants and microorganisms.  Pyruvate from glycolysis is converted by fermentation to lactate using the enzyme lactate dehydrogenase and the coenzyme NADH in lactate fermentation.  Alternatively it is converted to acetaldehyde and then to ethanol in alcoholic fermentation  Regulation of Glycolysis The rate of conversion of glucose into pyruvate is regulated to meet two major cellular needs:  Production of ATP  Provision of building blocks for synthetic reactions, such as the formation of fatty acids. Glycolysis can be regulated by three different types of mechanisms: 1. Changes in the rate of enzyme synthesis, Induction/ repression. (Transcription) 2. Hormonal control (glucagon and insulin) 3. Allosteric modification. Allosteric inhibition: Binding of a ligand outside the catalytic site—can activate OR inhibit Enzyme with Enzyme with Enzyme with “Simple” Enzyme Allosteric Allosteric Allosteric activation activating site inhibitory site and inhibitory sites Allosteric Allosteric Substrate Activator Inhibitor  Regulation of Glycolysis The reactions which are regulatory steps in glycolysis are: 1. Formation of glucose-6-phosphate from glucose (Hexokinase/glucokinase). 2. Fructose-6-phosphate to Fructose 1,6- bisphosphate (Phosphofructose kinase). 3. Formation of pyruvate from phosphoenol-pyruvate (Pyruvate kinase).  Regulation of Glycolysis- PFK-1  Phosphofructokinase (PFK-1) is the most important/ key regulatory enzyme in glycolysis which catalyses a rate limiting committed step (irreversible). 1. ATP/ADP-AMP  PFK is allosterically inhibited by ATP but this inhibition is reversed by ADP/AMP. This allows glycolysis to be responsive to the energy needs of the cell, speeding up when ATP is in short supply (and ADP/AMP is plentiful) so that more ATP can be made, and slowing down when sufficient ATP is already available. 2. Citrate  PFK-1 is also inhibited by citrate, the first product of the citric acid cycle.  A high level of citrate signals that there is a plentiful supply of citric acid cycle intermediates already and hence no additional breakdown of glucose via glycolysis is needed.  Regulation of Glycolysis - PFK-1 3. H+ ions  PFK is inhibited by H+ ions and hence the rate of glycolysis decreases when the pH falls significantly.  This prevents the excessive formation of lactate under anaerobic conditions and hence prevents the medical condition known as acidosis (a deleterious drop in blood pH).  Regulation of Glycolysis - PFK-1 4. Fructose 2,6-bisphosphate  A small amount of F-6-P is also diverted into an alternative pathway of regulation.  It reacts with an enzyme complex (binuclear complex) known as phosphofructokinase 2 (PFK2) and fructose bisphosphatase 2 (FBPase2).  Phosphofructokinase 2 (PFK2) converts F-6-P into F-2,6-Bisphosphate (powerful regulator of PFK-1).  F-2,6-BP in turn strongly activates PFK-1 and hence stimulates glycolysis. The overall effect is that when fructose 6-phosphate levels are high, PFK (and hence glycolysis) is stimulated.  F-2,6-BP is hydrolyzed back to fructose 6-phosphate by fructose bisphosphatase 2 (FBPase2).  PFK-2 and FBPase2 are highly regulated by the hormones glucagon and insulin  When glucose levels are high, the body needs to undergo glycolysis. Thus PFK-2 will be stimulated (converts F-6-P into F-2,6- Bisphosphate which stimulates PFK-1, resulting in F-1,6-BP).  Also, FBPase2 must be inhibited. How?  Glucagon phosphorylates both the enzymes  When PFK-2 is phosphorylated by glucagon, it is inhibited  When FBPase2 is phosphorylated by glucagon, it is activated  Insulin does the reverse by dephosphorylating the two enzymes.  When PFK-2 is dephosphorylated by insulin, it is activated  When FBPase2 is dephosphorylated by glucagon, it is inhibited.  Regulation of Glycolysis - Hexokinase  Hexokinase, which catalyses the first irreversible step of glycolysis, is inhibited by glucose 6-phosphate.  Thus when PFK is inhibited, fructose 6-phosphate builds up and so does glucose 6-phosphate since these two metabolites are in equilibrium via phosphogluco-isomerase.  The hexokinase inhibition then reinforces the inhibition at the PFK step.  Glucose 6-phosphate, the product of the hexokinase reaction, can also feed into glycogen synthesis or the pentose phosphate pathway.  Thus the first irreversible step that is unique to glycolysis is that catalysed by PFK and hence this is the main control step.  Regulation of Glycolysis - Pyruvate kinase (PK)  Is inhibited by ATP, long chain fatty acyl co-A and Acetyl co-A  Glucagon acts as a hormonal inhibitor of pyruvate kinase by phosphorylating it.  Thus, Pyruvate kinase is active in its dephosphorylated state and inactive in phosphorylated state.  Pyruvate kinase is activated by Fructose 1, 6-bisphosphate. (Feed forward reaction FFR).  Insulin dephosphorylates PK thereby activating it.  Summary of glycolysis  Glycolysis is a near-universal pathway by which a glucose molecule is oxidized to two molecules of pyruvate, with energy conserved as ATP and NADH.  All ten glycolytic enzymes are in the cytosol, and all ten intermediates are phosphorylated compounds of three or six carbons.  In the preparatory phase of glycolysis, ATP is invested to convert glucose to fructose 1,6-bisphosphate. The bond between C-3 and C- 4 is then broken to yield two molecules of triose phosphate.  Summary of glycolysis  In the payoff phase, each of the two molecules of glyceraldehyde 3- phosphate derived from glucose undergoes oxidation at C-1; the energy of this oxidation reaction is conserved in the formation of one NADH and two ATP per triose phosphate oxidized. The net equation for the overall process is;  Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP. Hexokinase, PFK-1, and pyruvate kinase are all subject to allosteric regulation that controls the flow of carbon through the pathway and maintains constant levels of metabolic intermediates.